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Abstract

Background

Hyperhomocysteinemia (HHcy) causes increased oxidative stress and is an independent
risk factor for cardiovascular disease. Oxidative stress is now believed to be a major
contributory factor in the development of non alcoholic fatty liver disease, the most
common liver disorder worldwide. In this study, the changes which occur in homocysteine
(Hcy) metabolism in high fat-diet induced non alcoholic fatty liver disease (NAFLD)
in rats were investigated.

Methods and results

After feeding rats a standard low fat diet (control) or a high fat diet (57% metabolisable
energy as fat) for 18 weeks, the concentration of homocysteine in the plasma was significantly
raised while that of cysteine was lowered in the high fat as compared to the control
diet fed animals. The hepatic activities of cystathionine β-synthase (CBS) and cystathionine
γ-lyase (CGS), the enzymes responsible for the breakdown of homocysteine to cysteine
via the transsulphuration pathway in the liver, were also significantly reduced in
the high fat-fed group.

Conclusions

These results indicate that high fat diet-induced NAFLD in rats is associated with
increased plasma Hcy levels caused by down-regulation of hepatic CBS and CGL activity.
Thus, HHcy occurs at an early stage in high fat diet-induced NAFLD and is likely to
contribute to the increased risk of cardiovascular disease associated with the condition.

Keywords:

Background

Non alcoholic fatty liver disease (NAFLD) is the most common liver disorder in the
world, and in obesity, type 2 diabetes and related metabolic diseases, its incidence
reaches 70-90% [1]. The disease is characterised by the accumulation of triacylglycerol (TG) inside
liver cells, and the condition can progress into more serious liver disease, such
as non alcoholic steatohepatitis (NASH), liver fibrosis, cirrhosis, and more rarely,
liver carcinoma [1]. Although it is known that progression of the disease is more likely to occur in
patients with metabolic diseases [2], the factors involved are not well understood. However, oxidative stress coupled
with insulin resistance is believed to play an important role [3].

Current evidence indicates that insulin resistance causes oxidative stress via increased
oxidation in the liver, raised formation of reactive oxygen species, higher levels
of hepatic lipid peroxidation, protein oxidation and pro-inflammatory cytokine production
and decreased antioxidant capacity in the plasma [3-7]. Since the hepatic steatosis in NAFLD is believed to follow from the development
of insulin resistance, oxidative stress is now considered to be one of the major causes
of NAFLD [8,9].

Recent studies have suggested that NAFLD is associated with accelerated atherosclerosis
[10], but the underlying mechanisms are not well understood. Moderately raised plasma
levels of homocysteine (Hcy) have been found to be associated with atherosclerosis
development, and hyperhomocysteinemia (HHcy) is considered to be an independent risk
factor for cardiovascular disease [11,12]. Hcy is a thiol-containing amino acid involved in the metabolism of methionine in
the liver. Methionine from dietary sources is converted to S-adenosyl methionine (SAM)
by methionine adenosyl transferase (MAT). SAM is the methyl donor in most biological
methylation reactions, and various methyltransferases (eg phosphatidyl-ethanolamine
N-methyltransferase (PEMT) and glycine N-methyltransferase (GNMT)) are involved in
its usage for the formation of phospholipids, myelin, and other macromolecules [12]. The product of the methyl transfer reactions is S-adenosyl homocysteine (SAH), which
is then hydrolyzed to Hcy. Once formed, Hcy may be either remethylated to methionine
(methionine cycle) or metabolised to cysteine (Cys) in a two step pathway catalysed
by the enzymes cystathionine β-synthase (CBS) and cystathionine γ-lyase (CGL) (the
transsulphuration pathway) [12,13]. HHcy is known to be associated with atherosclerosis and other pathologies, and oxidative
stress, due to NADPH oxidase or NO synthase dependent generation of superoxide anion
combined with a decrease in antioxidant enzyme activity, is thought to be a major
factor in its effects [11,12]. In particular, when HHcy is due to disorders of the transsulphuration pathway, it
associates with a reduced supply of Cys for the formation of the antioxidant glutathione
(GSH) [13]. It has been reported that HHcy caused by CBS deficiency leads to disturbances in
the regulation of lipid metabolism and fat accumulation in the liver [14,15]. Moreover, plasma Hcy is elevated in patients with NAFLD, and is a predictor of steatohepatitis
[16].

Our previous work has shown that feeding rats a high fat diet (57% of energy from
fat) induces insulin resistance, hypertriglyceridemia, hepatic steatosis and liver
damage, which are characteristic of NAFLD and thus provides a suitable model for the
early stages of the disease [17,18]. In the present study, we have used this model to test the hypothesis that high fat
diet-induced NAFLD in rats modifies hepatic Hcy metabolism via modulation of the transsuphuration
pathway.

Methods

Animals and diets

Male Wistar rats (200 g) (Harlan, S. Pietro al Natisone, Italy) were housed individually
and allowed food and water ad libitum. All procedures conformed to the Guidelines of the European Community Council for
animal care and use and to Decree 116/92, the Italian enforcement of the European
Directive 86/609/EEC. The experimental protocol was approved by the Animal Care Ethics
Committee of the Istituto Superiore di Sanità (ISS) and, according to the national
law requirement the communication of the study has been sent by the ISS to the Animal
Welfare Office ( Office VI) of the Italian Minister of Health (Communication Protocol
Number 983/SSA/07). Rats were fed a standard low fat diet (4.3% fat, 10% of the metabolizable
energy; control diet, 6 rats) or a diet containing 35% fat (31.6% saturated fat and
3.2% unsaturated fat, 57% of the metabolizable energy [18]; high fat diet, 4 rats) (Mucedola srl, Settimo Milanese, Italy) for 18 weeks. All
rats were then sacrificed after fasting overnight. Blood samples were collected via
heart puncture with the rats under terminal anesthesia (Acepromazine 2.5 mg/kg + Xylazine
1 - 5 mg/kg im) and centrifuged (3,500 rpm, 15 min, 6°C) to obtain the plasma. Livers
were excised, washed with cold physiological saline (0.9%), samples (200 mg) were
homogenised in methanol (5 ml) and the lipids extracted [19].

Results and discussion

Results

Liver TG concentrations were increased by about 2.6 fold in the rats fed the high
fat diet as compared to the control diet and there was a more modest, but significant
increase (+30%) in liver cholesterol levels (Table 2). In rats fed the high fat diet plasma insulin levels were about 6 fold higher than
in animals given the control diet (Figure 1A) and the HOMA-IR showed a rise of a similar magnitude (Figure 1B). Plasma glucose levels, however, were not significantly changed (control diet, 8.75
± 0.70 mmol/l; high fat diet, 8.45 ± 0.27 mmol/l).

Figure 1.Effect of high fat diet feeding on plasma insulin and HOMA-IR. Rats were fed standard low fat diet (Control diet) or a high fat diet for 18 weeks.
Blood samples were then collected and the concentration of insulin in plasma (A) were
determined and the HOMA-IR (B) calculated. Data are the mean from 6 (Control diet)
or 4 (High fat diet) animals and error bars show the SEM. **P < 0.01, ***P < 0.001
vs Control diet.

Plasma Hcy and Cys concentrations in control diet- and high fat diet -fed rats are
shown in Figure 2. Hcy concentrations in the plasma of the rats fed the high fat diet were significantly
higher than those in control animals (+31%, P < 0.05) (Figure 2A), and this increase was accompanied by a decrease in plasma Cys levels (-15%, P <
0.001) (Figure 2B). In addition, the activities of both CBS and CGL in the liver were significantly
reduced by high fat feeding (Figure 2). CBS activity was decreased by about 15% (P < 0.05) (Figure 2C), while that of CGL was lowered by approximately 23% (P < 0.05) (Figure 2D).

Determination of the relative abundance of mRNA transcripts for MAT1A, PEMT and GNMT
showed no significant changes between the control and high fat-fed groups (Table 3).

Table 3. Effect of high fat diet feeding on expression of mRNA for methyltransferases

Discussion

Oxidative stress is now believed to be an important factor in the development of NAFLD
[3-5] acting as a cause as well as a consequence of hepatic steatosis [6,25]. Here, we investigated the changes which occur Hcy metabolism in a high fat-diet
induced model of NAFLD in rats. The high fat diet used caused an increase in liver
TG ( × 2.6) and cholesterol (+ 30%) and plasma insulin levels and the HOMA-IR were
increased by 6 fold (Figure 1). These results are in agreement with our previous more extensive characterisation
of the effects of this diet in inducing NAFLD [17], and indicate that the animals are a good model for the condition.

A number of studies have implicated HHcy in premature atherosclerosis development,
and oxidative stress is thought to be a major factor in this effect [11,12]. NAFLD is associated with accelerated atherosclerosis [10], and oxidative stress is now believed to be an important factor in the development/progression
of the condition [3-5]. Recent studies have indicated that increased oxidative stress is an important trigger
for insulin resistance, which is believed to cause the disturbances in liver lipid
metabolism that result in NAFLD. The resulting fat accumulation is then thought to
promote further production of reactive oxygen species, thus oxidative stress can be
considered to be both a cause and a consequence of hepatic steatosis [1,6,25]. In the liver, Hcy is produced from SAH formed during methyl transfer reactions involving
SAM and methyltransferase enzymes such as GNMT and PEMT, and removed by conversion
to Cys via the transsulphuration pathway which involves CBS and CGL [11-13]. Although it has been shown that the plasma Hcy levels are elevated in alcoholic
fatty liver disease [26] and that HHcy is associated with the development of hepatic steatosis in mice deficient
in CBS and in human subjects with a genetic defect in the enzyme [14-16,27], few studies have investigated Hcy metabolism in NAFLD. Gulsen et al. [16] have reported that plasma Hcy concentrations were significantly higher in NAFLD patients
as compared to healthy subjects, and that they were a good predictor of progression
to NASH. In contrast, however, another study found no significant difference in plasma
Hcy levels in obese subjects with or without NAFLD [28].

The results of our experiments show that plasma Hcy is significantly increased in
high fat diet-induced NAFLD in rats, and that this change is accompanied by a decrease
in plasma Cys (Figure 2A,B), suggesting that the transsulphuration pathway is affected. Determination of the
activities of the two enzymes of the pathway showed that both are significantly down-regulated
in high fat diet-induced NAFLD (Figure 2C,D). We found no evidence, however, for changes in the expression of other key enzymes
in the hepatic methionine cycle, including MAT1A, the gene which encodes the catalytic
subunit of the isoenzymes MATI and MATIII which are expressed in adult liver [29], and the methyltransferases GNMT and PEMT (Table 3). Since these enzymes are methyltransferases while CBS and CGL are lyases, it is
likely that they are regulated by different mechanisms. In addition, we have only
measured mRNA expression for these enzymes, thus we cannot rule out the possibility
that there may be post transcriptional changes which modulate their activity in response
to high fat feeding. In contrast to our results, Kwon et al. [30] have reported that CBS activity was unchanged in rats in which NAFLD was induced
by feeding a diet containing 71% of the energy as fat, while CGL activity was increased
by about 35%. In this study, however, the control diet contained 35% of energy from
fat, which is considerably higher than the 10% of energy from fat in our control diet.
Thus, our findings indicate that high fat diet-induced NAFLD is associated with HHcy,
and that this is caused by reduced conversion to Cys via the transsulphuration pathway,
while the expression of methyltransferases involved in liver methionine metabolism
is not changed. As it has been estimated that as much as 50% of the Cys required for
GSH synthesis is formed from Hcy via this route, and the availability of Cys is a
limiting factor for GSH production [12,31], in the long term this may lead to a decrease in body levels of this antioxidant.

Conclusions

In conclusion, these results reported here show that high fat diet-induced NAFLD in
rats causes HHcy and that this is due to down-regulation of hepatic CBS and CGL activity.
Thus, HHcy is an early feature of high fat diet-induced NAFLD and is likely to contribute
to the increased risk of cardiovascular disease associated with the condition.

Competing interests

The authors declare that they have no competing interests.

Authors' contributions

PA, XB, BR, AC, MN carried out experimental work; SP, EB, BO, KMB, designed and planned
the study, and also drafted and critically revised the manuscript. All the authors
contributed to the interpretation and discussion of results related to their part
of the work and read and approved the final manuscript.

Acknowledgements

We thank Dr Fiorella Ciaffoni and Dr Loretta Diana for their technical support. We
are grateful to Mr. Antonio Di Virgilio and Mr. Agostino Eusepi for their skilled
help with animal work. Our gratitude to Dr. Giorgio Borioni for his useful comments.
The work was partially supported by the Ricerca Finalizzata of the Italian Ministry
of Health (Ref ISS R26).